This thesis explored the possibility of using synthetic Ras peptides to stim ulate specific Th and CTL responses against an oncogenic m utation at position 12 of p21 Ras proteins (C hapter 3 and 4). Clearly in som e m ouse strain s m u ta n t Ras p e p tid e could in d u ce Th cells w h ic h specifically recognised the m u ta n t pep tid e and protein. These results suggested th at u n d e r certain circum stances tu m o u r related a m utation in p21Ras protein could be im m unogenic and probably m u tan t Ras peptides could be used to b o o st m u tatio n -sp ecific im m u n e responses. R ecently sev eral research groups have reported sim ilar findings (Jung an d Schluesener, 1991; Peace, et al., 1991; Gedde-Dahl, et al., 1992a; Gedde-Dahl, et al., 1992b). One distinct point w e m ade in this study w as th at m u tan t Ras peptide induced Th cells in H-2^ mice also recognised norm al Ras peptide. Since w e d id not analyse m any m ouse strains, it is difficult to tell how frequently Th cells cross react to m u tan t an d norm al Ras peptides. H ow ever, it is a reasonable thinking th at peptide vaccine should be designed according to each MHC haplotype.
The analysis of CTL resp o n ses ag ain st sy n th etic Ras p e p tid es bro u g h t us into a different field from o u r initial interest w hich w as to find a targ et for tu m o u r im m unotherapy. In three m ouse strains analysed w e d id not get CTL w hich specifically recognised glycine to valine m utation at position 12 of Ras peptides. H ow ever, Ras peptides in duced CTL show ed very interesting features in th at they recognised Ras p ep tid es in a MHC unrestricted fashion. U nfortunately w e have n o t m anaged to identify the real epitope recognised by these CTL. It w ill be very interesting to know w h at the size of this special CTL epitope is. It is possible th at these MHC unrestricted CTL recognised 8 or 9 m er peptides like m ost of classical MHC restricted CTL do. A lternatively they m ight recognise longer peptides as w e show ed in one experim ent that 18 m er Ras peptide could n o t sensitise CTL lysis. P resently w e are try in g to identify th e n a tu re of the m inor c o m p o n e n ts re s p o n s ib le fo r M H C u n r e s tric te d CTL re s p o n s e s. Identification of the p ep tid e seqeunce w ould help us to u n d e rstan d the m echanism of M HC unrestricted CTL recognition.
O nce th e Ras CTL ep ito p e h as b een id e n tifie d , it w o u ld be in te restin g to determ ine: I) do M HC class I m olecules b in d th e Ras p ep tid es in a sim ilar w ay as classical p ep tid e antigens, i.e. b in d in g the p e p tid e s in th e groove? This can p o ssib ly be a d d re sse d b y p e p tid e com petition assays. W e h ad som e p rim ary d ata sh o w in g th at th e CTL
recognition of Ras peptides could not be inhibited by a T cell epitope w ith a high affinity for binding to class I molecules. II) w hich dom ain of class I m olecules is critical for b inding Ras peptides? This can be an sw ered b y using cells transfected w ith chemeric class I m olecules as APC to p resen t Ras peptides. IE) w hich p a rt of TCR is involved in the CTL recognition of Ras f)eptides? M ore anti-TCR m A b should be u sed to analyse extensively the TCR usage of long term Ras p ep tid e-sp ed fic CTL lines an d clones. Also, anti-TCR m Ab can be applied to block the CTL recognition in CTL a ssa y s.
U sing synthetic p ep tid es, especially longer p ep tid es, for T cell epitope m ap p in g can be very m isleading. The lesson w e learned w as th at to be sure w ith the T cell epitope being analysed, HPLC separated fractions of the synthetic peptides should be tested. A ttention should also be p aid to p e p tid e syn th esis. As sh o w n in o u r stu d y in d e p e n d e n t sy sth e sise d peptides can be biologically different.
There are a lot of questions w hich need to be answ ered about m H antigens m ediated graft rejection and GVHD. T cell recognition of HPLC p u rifie d cellular p ep tid es is a very useful tool w hich can h elp u s to u n d e rs ta n d th e m olecular biology of m H an tig en s. T he n u m b e r of im m unodom inant m H peptides can be revealed by using this technique. We did n o t analyse the peptide recognition by m H antigen-specific T h cells in this study. According to Roopenian's proposal one m H locus consists of a t lea st tw o genes en co d in g Th a n d CTL e p ito p e s in d e p e n d e n tly
(R oopenian, 1992). M ost likely th e tw o d ifferen t e p ito p es co u ld be sep arated by a HPLC colum n w ith high resolution capacity. It w ill be interesting to com pare the p ep tid e recognition p attern s of m H antigen- spedfic T h cells and CTL.
U sing HPLC purification technique to achieve m icrosequence of m H pep tid es definately is n o t an easy job. A lthough w e u sed m ore th an 200 mice, w e have not yet succeeded to obtain the m icrosequence. To reach this goal, w e probably need to use better equipm ents w hich can m inim ise the loss of m aterial and maximise the sensitivities of detection.
REFERENCES
A cha-O rbea, H., A. N. Shakhov, L. Scarpellino, E. Kolb, V. M uller, S. A. Vessaz, R. Fuchs, K. Blochlinger, P. Rollini, J. Billotte and a. 1. et. (1991). C lonal d e le tio n of V b eta 14-bearing T cells in m ice tran sg en ic for m am m ary tu m o u r virus. Nature. 350:207.
A dorini, L., S. J. Ullrich, E. A ppella an d S. Fuchs. (1990). Inhibition by brefeldin A of presentation of exogenous protein antigens to M HC class Ti res tricted T cells. Nature. 346:63.
Allison, J. P. an d W. L. H avran. (1991). The im m unobiology of T cells w ith in v a ria n t gam m a d elta an tig en receptors. A n n u Rev Immunol.
9:679.
Am it, A. G., R. A. M ariuzza, S. E. Phillips and R. J. Poljak. (1986). Three- dim ensional structure of an antigen-antibody complex at 2.8 A resolution.
Science. 233:747.
A nderson, K., P. Cresswell, M. G am m on, J. H erm es, A. W illiam son and H. Z w ee rin k . (1991). E n d o g en o u sly sy n th e siz e d p e p tid e w ith an en d o p lasm ic reticu lu m signal sequence sensitizes an tig en p rocessing m u tan t cells to class I-restricted cell-mediated lysis. / Exp Med. 174:489.
A ndo, K., I. N akashim a, F. Nagase, K. Isobe, K: K aw ashim a, Y. H asegaw a, T. Y oshida, T. Iw am oto, T. H asegaw a, Y. M uro a n d a. 1. et. (1988). In d u ctio n a n d ch aracterizatio n of m in o r h isto co m p atib ility antigens. Specific p rim ary cytotoxic T lym phocyte responses in vitro. / Immunol.
140:723.
Anichini, A., G. Fossati and G. Parm iani. (1987). Clonal analysis of the cytolytic T-cell response to hum an tum ors. Immunol. Today. 8:385.
A rnold, D., J. Driscoll, M. A ndrolew icz, E. H ughes, P. C ressw ell and T. Spies. (1992). Proteasom e subunits encoded in the MHC are not generally required for the processing of peptides b o u n d by M HC class I molecules.
A ttaya, M., S. Jameson, C. K. M artinez, E. Hermel, C. Aldrich, J. Form an, K. F. Lindahl, M. J. Bevan and J. J. Monaco. (1992). Ham -2 corrects the class I antigen-processing defect in RMA-S cells. Nature. 355:647.
Bailey, D. W. and L. E. M obraaten. (1969). Estimates of the num ber of loci contributing to the histoincom patiibility betw een C57BL/6 an d BALB/c strains of mice. Transplantation. 7:394.
Bailey, D. W. (1975). Genetic of histocom patibility in mice. I. N ew lo d and congenic lines. Immunogenetics. 2:249.
Barbacid, M. (1987). ras genes. Ann. Rev. Biochem. 56:779.
Bamd, D. L., M. S. Lan, R. S. M etzgar and O. J. Finn. (1989). Specific, major h istocom patibility com plex-unrestricted recognition of tum or-associated m ucins by hum an cytotoxic T cells. Proc Natl Acad Sci U S A . 86:7159.
Benichou, G., P. A. Takizaw a, P. T. H o, C. C. Killion, C. A. O lson, M. McMillan and E. E. Sercarz. (1990). Im m unogenicity and tolerogenicity of self-major histocom patibility complex peptides. / Exp Med. 172:1341.
Berzofsky, J. A. (1991). M echanisms of T cell recognition w ith application to vaccine design. Mol Immunol. 28:217.
Bevan, M. J. (1975). The m ajor histocom patibility com plex determ ines susceptibility to cytotoxic T cells directed against m inor histocom patibility antigens. /. Exp. Med. 142:1349.
Bierer, B. E., B. P. Sleckman, S. E. Ratnofsky an d S. J. Burakoff. (1989). The biologic roles of CD2, CD4, and CD8 in T-cell activation. A n n . Rev. Im m unol. 7:579.
Bjorkman, P. J., M. A. Saper, B. Sam ouroui, W. S. Bennett, J. L. Strom inger a n d D. C. W iley. (1987a). S tru c tu re of th e h u m a n class II histocom patibility antigen, HLA-A2. Nature. 329:506.
Bjorkman, P. J., M. A. Saper, B. Sam raoui, W. S. Bennett, J. L. Strom inger an d D. C. W iley. (1987b). The foreign antigen b in d in g site an d T cell recognition regions of class I histocom patibility antigens. Nature. 329:512.
Blackman, M., J. K appler, and P. M arrack. (1990). The role of the T cell receptor in positive an d negative selection of developing T cells. Science.
248:1335.
Blackman, M. A., F. E. Lund, S. Surm an, R. B. Corley and D. L. W oodland. (1992). M ajor h isto co m p atib ility c o m p lex -restricted re c o g n itio n of retroviral superantigens by V beta 17+ T cells. / Exp Med. 176:275.
Bluestone, J. A., R. Q. Cron, T. A. Barrett, B. H oulden, A. I. Sperling, A. D ent, S. H edrick, B. R ellahan a n d L. A. M atis. (1991). R ep erto ire dev elo p m en t an d ligand specificity of m u rin e TCR gam m a delta cells.
Imm unol Rev. 120:5.
Blum, J. S. and P. Cresswell. (1988). Role for intracellular proteases in the processing an d tran sp o rt of class II HLA antigens. Proc. Natl. Acad. Sci. USA. 85:3975.
Bodm er, H. C., F. M. Gotch and A. J. McMichael. (1989). Class I cross restricted T cells reveal low resp o n d er allele d u e to processing of viral antigen. Nature. 337:653.
Bos, J. L. (1989). ras oncogenes in hum an cancer: a review . Cancer Res.
49:4682.
Brodsky, F. M. an d L. E. Guagliardi. (1991). The cell biology of antigen processing and presentation. Annu Rev Immunol. 9:707.
B rodsky, F. M. (1992). A ntigen processing an d p resen ta tio n : close encounters in the endocytic pathw ay. Trends in Cell Biol. 2:109.
Brown, J. H., T. Jardetzky, M. A. Saper, B. Sam raoui, P. J. Bjorkman an d K. C. Wiley. (1988). A hypothetical m odel of the foreign antigen binding site of class n histocom patibility molecules. Nature. 332:845.
B row n, M. G., J. D riscoll and J. J. M onaco. (1991). S tru ctu ral an d serological sim ilarity of M HC-linked LMP an d proteasom e (m ulticatalytic proteinase) complexes. Nature. 353:355.
C arb o n e, F. R., M. W. M oore, J. M. Sheil an d M. J. Bevan. (1988). Induction of cytotoxic T lym phocytes by prim ary in vitro stim ulation w ith peptides. / Exp Med. 167:1767.
C arlsso n , R., H. Fischer an d H. O. Sjogren. (1988). B inding of staphylococcal enterotoxin A to accessory cells is a req u irem en t for its ability to activate hum an T cells. J Immunol. 140:2484.
Cazenave, P. A., P. N. Marche, M. E. Jouvin, D. Voegtle, F. Bonhomme, A. Bandeira an d A. Coutinho. (1990). V beta 17 gene polym orphism in w ild- d eriv ed m ouse strains: tw o am ino acid su b stitu tio n s in th e V beta 17 region greatly alter T cell receptor specificity. Cell. 63:717.
C eru n d o lo , V., J. A lexander, K. A nderson, C. Lam b, P. C ressw ell, A. M cM ichael, F. Gotch a n d A. T ow nsend. (1990). P resen tatio n of viral an tig en controlled by a gene in the m ajor h istocom patibility complex.
Nature. 345:449.
C eru n d o lo , V., T. Elliott, J. Elvin, J. Bastin, H. G. R am m ensee an d A. Tow nsend. (1991). The binding affinity an d dissociation rates of peptides for class I m ajor histocom patibility com plex m olecules. Eur ] Immunol.
21:2069.
C hatila, T. and R. S. Geha. (1992). Superantigens. Curr Opin Immunol.
4:74.
C hen, B. P., A. M adrigal an d P. P arham . (1990). C ytotoxic T cell recognition of an endogenous class I HLA peptide p resen ted by a class II HLA molecule. / Exp Med. 172:779.
Chicz, R. M., R. G. U rban, W. S. Lane, J. C. Gorga, L. J. Stem , D. A. Vignali an d J. L. Strom inger. (1992). P redom inant n atu rally processed peptides b o u n d to H LA -DRl are derived from M H C -related m olecules a n d are heterogeneous in size. Nature. 358:764.
Choi, Y. W., A. H erm an, D. DiGiusto, T. W ade, P. M arrack an d J. Kappler. (1990). R esidues of the variable region of the T-cell-receptor beta-chain th at interact w ith S. aureus toxin superantigens. Nature. 346:471.
Choi, Y., J. W. Kappler and P. Marrack. (1991). A super antigen encoded in the open reading fram e of the 3* long term inal rep eat of m ouse m am m ary tu m o u r virus. Nature. 350:203.
C hristinck, E. R., M. A. Luscher, B. H. Barber and D. B. W illiams. (1991). P e p tid e b in d in g to class I M HC o n liv in g cells an d q u a n tita tio n of complexes required for CTL lysis. Nature. 352:67.
C olom bo, M. P., R. Jaenisch and P. J. W ettstein. (1987). E ndogenous re tro v iru se s lead to th e ex p ressio n of a h isto co m p atib ility an tig en detectable by skin graft rejection. Proc Natl Acad Sci U S A . 84:189.
Cox, J. H., J. W. Yewdell, L. C. Eisenlohr, P. R. Johnson and J. R. Bennink. (1990). A ntigen presentation requires transport of M HC class I molecules from the endoplasm ic reticulum . Science. 247:715.
Cresswell, P. (1992). C hem istry and functional role of the invariant chain.
Curr Opin Immunol. 4:87.
Dausset, J. (1958). Iso-leuco-anticorps. Acta Haematol. 20:156.
D avidson, H. W., P. A. Reid, A. Lanzavecchia a n d C. W atts. (1991). Processed antigen binds to new ly synthesized MHC class II m olecules in antigen-specific B lymphocytes. Cell. 67:105.
Davis, A. P. and D. C. Roopenian. (1990). Com plexity at the m ouse m inor histocom patibility locus H-4. Immunogenetics. 31:7.
Davis, M. M. and P. J. Bjorkman. (1988). T-cell antigen receptor genes and T-cell recognition. Nature. 334:395.
de Bueger, M., F. Verreck, E. Blokland, J. W. D rijifhout, R. A m ons, F. Koning and E. Goulmy. (1993). Isolation of an HLA-A2.1 extracted hu m an m inor histocom patibility peptide. Eur. J. Immunol. 23:614.
D egen, E. an d D. B. W illiams. (1991). P articipation of a novel 88-kD pro tein in the biogenesis of m urine class I histocom patibility m olecules. /
Cell Biol. 112:1099.
Dellabona, P., J. Peccoud, J. Kappler, P. Marrack, C. Benoist an d D. M athis. (1990). Super antigens interact w ith MHC class II molecules outside of the antigen groove. Cell. 62:1115.